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PARP-1 modulation of mTOR signaling in response to a DNA alkylating agent

Chantal Ethier1, Maxime Tardif, Laura Arul

  • 1Cancer Axis, CHUQ Research Center and Faculty of Medicine, Laval University, Quebec City, Quebec, Canada.

Plos One
|October 31, 2012
PubMed

Insights

Poly(ADP-ribose) polymerase-1 (PARP-1) activation triggers necrotic cell death by altering cellular energy and inhibiting mTOR signaling. Antioxidants and PARP inhibitors block these PARP-1-mediated events.

Area of Science:

  • Cell Biology
  • Biochemistry
  • Molecular Biology

Background:

  • Poly(ADP-ribose) polymerase-1 (PARP-1) plays a crucial role in cellular responses to DNA damage, influencing cell death pathways like autophagy, apoptosis, and necrosis.
  • The specific cell death outcome following PARP activation is dependent on injury severity and cell type.

Purpose of the Study:

  • To investigate the molecular mechanisms by which PARP-1 activation leads to necrotic cell death in HEK293 cells exposed to N-methyl-N'-nitro-N'-nitrosoguanine (MNNG).
  • To elucidate the role of altered cellular energy levels and the mTOR signaling pathway in PARP-1-mediated necrosis.

Main Methods:

  • HEK293 cells were treated with MNNG, an alkylating agent, to induce PARP-1 activation.
  • Levels of NAD+, ATP, and AMP were measured to assess cellular energy status.
  • Activation of AMP-activated protein kinase (AMPK) and modulation of the mTORC1 and mTORC2 pathways were analyzed through specific protein phosphorylation.
  • The effects of PARP-1 inhibition (using AG14361) and antioxidant treatment (N-acetyl-L-cysteine, NAC) were evaluated.

Main Results:

  • MNNG exposure led to massive poly(ADP-ribose) (PAR) synthesis, decreased NAD+ and ATP, and increased AMP levels.
  • AMPK activation and mTORC1 inhibition (via Raptor phosphorylation) occurred, alongside decreased S6 phosphorylation.
  • Loss of Akt phosphorylation at Ser473 preceded cell death by necrosis.
  • Inhibition of PARP-1 or treatment with NAC abrogated all observed signaling events and prevented necrosis.

Conclusions:

  • PARP-1 activation and subsequent PAR synthesis significantly impact cellular energy homeostasis and inhibit mTORC1 signaling.
  • Modulation of the mTORC2 complex, evidenced by Rictor and Akt phosphorylation changes, is implicated in PARP-1-induced necrosis.
  • Reactive oxygen species (ROS) production is involved in PARP-1 activation and the subsequent modulation of mTOR signaling pathways.
  • PARP-1 and PAR play integral roles in orchestrating necrotic cell death through the intricate balance of cellular signaling pathways.

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